Project Summary/Abstract Disruption of barrier function at the ocular surface epithelia is associated with a wide range of disorders that includes dry eye?an age-related disease affecting millions of people worldwide and whose pharmacological treatment remains unresolved. Terminally differentiated stratified epithelial cells in cornea and conjunctiva maintain barrier function through a specialized protective structure composed of transmembrane mucins, a group of heavily glycosylated proteins characterized by extremely large extracellular domains. Galactosyl residues on mucin glycans are cross-linked on the apical glycocalyx by the multimeric protein galectin-3 to prevent cellular damage. Critical to preventing the decline in cellular function and homeostasis is the hexosamine pathway?a series of anabolic reactions that generate increased synthesis of N-glycan precursors. Activation of the hexosamine pathway leads to increased branching of N-glycans in the medial Golgi and the formation of cell-surface galectin-3 lattices that can modulate cell differentiation. Importantly, metabolic supplementation with hexosamine pathway metabolites is also known to enhance protein quality control mechanisms. Despite these critical roles, the relevance of N-glycosylation and the function of hesoxamine metabolites in ocular surface health and disease remains understudied. We hypothesize that N- glycans have a dynamic role in ocular surface epithelial cells, changing in response to inflammation and driving mucin barrier and stress responses. The long-term objective of this proposal is to determine the contribution of the N-glycan branching in promoting ocular surface health, and whether activation of the hexosamine pathway can be used for therapeutic gain in the eye. The following specific aims will address this objective: (1) to characterize mucin N-glycans in human corneal epithelial cells and their relationship to galectin-3 under normal and pro-inflammatory conditions, (2) to determine the regulatory role of N-glycans in promoting barrier function and reducing stress responses at the ocular surface, and (3) to evaluate whether activation of the hexosamine pathway promotes ocular surface homeostasis. This research will address the largely unstudied function of N-glycans in ocular surface barrier function and stress responses, and explore the potential of pharmacologically activating the hexosamine pathway for the treatment of ocular surface diseases.